Department of Biology and Biological Engineering, Chalmers University of Technology, SE41296 Gothenburg, Sweden.
Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE41296 Gothenburg, Sweden.
Proc Natl Acad Sci U S A. 2018 Nov 20;115(47):E11025-E11032. doi: 10.1073/pnas.1809921115. Epub 2018 Nov 5.
Baker's yeast is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.
面包酵母是用于重组蛋白生产的最重要且应用最广泛的细胞工厂之一。为了提高其蛋白生产能力，人们已经应用了许多策略来对该酵母进行工程改造，但生产力仍然相对较低，并且随着市场需求的增加，确定新的基因靶标非常重要，特别是与之前确定的靶标具有协同作用的靶标。尽管蛋白产量有所提高，但以前的研究很少关注与细胞内蛋白保留相关的过程。在这里，我们确定了与分泌和运输途径、组蛋白去乙酰化酶复合物以及碳水化合物代谢过程相关的遗传修饰作为提高酵母中蛋白分泌的靶点。除了增加蛋白分泌外，我们还发现在内体到高尔基体运输过程中的修饰可有效地减少蛋白保留。通过对几个新鉴定的基因靶标的组合遗传操作，我们通过分批补料培养将酵母的蛋白生产能力提高了五倍以上，最佳工程化菌株可以生产 2.5 g/L 的真菌α-淀粉酶，细胞内保留的重组蛋白不到 10%。
